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Notes: The authors examined the role of sxy expression in hypercompetence of Haemophilus influenza. The 1.8kb sxy gene was subcloned into the pALTER®-1 Vector and point mutations made using the Altered Sites® II in vitro Mutagenesis System. The mutated gene was sequenced and subcloned back into the original plasmid. The wildtype and mutant sxy genes were transcribed, dephosphorylated and labeled with 32P ATP. The end-labeled RNAs were then subjected to S1 nuclease mapping. The E. coli S30 Extract System for Linear Templates was used with the wildtype and mutant sxy constructs and the expression levels of the expressed protein measured by spot blotting. (3995)

Notes: The authors devised an mRNA display system to generate a peptide library with multiple nonnatural amino acids incorporated into the proteins, an important feature of peptide libraries for successful drug discovery. An mRNA with 3 four-base codons at a random position was used as a template in an in vitro translation system in the presence of charged tRNAs carrying four-base codons. In vitro translations were performed using 3.6 × 1013 molecules of mRNA template and the E. coli S30 Extract System. The mRNA template contained a T7 tag sequence, so the translation products could be detected using an anti-T7 tag antibody and the Anti-Mouse IgG (H+L), AP Conjugate. The mRNA-displayed peptides also incorporated a polyhistidine tag so that they could be purified using the MagneHis™ Ni-Particles. After selecting for the desired protein characteristic, the mRNA portion of the mRNA-displayed peptides was reverse transcribed and quantitated by real-time PCR. PCR products were cloned into the pGEM®-T Vector prior to sequencing. (3651)

Notes: In this paper, the effect of the novel antibiotic GS7128 on translation was determined in cell-free systems. The effective concentration for inhibition of translation was determined for both prokayotes and eukaryotes by translating β-galactosidase from a pGEM® vector in E. coli S30 extracts, and luciferase control RNA in rabbit reticulocyte lysates. It was determined that GS7128 partially inhibits initiation and can fully inhibit elongation of peptides by blocking the peptidyl transferase reaction. GS7128 was shown to bind ribosomes differently than any other characterized antibiotic. GS7128 resistant mutants were not resistant to any other antimicrobial agents. (2686)

Notes: To assay for the effect of experimental compounds on bacterial translation, the test compound was added to the control reaction of pBESTluc™ Control DNA and an E. coli S30 Extract System. It is not specified whether the circular or linear extract system is used. The inhibition was measured as a function of luciferase activity. (0864)

Notes: An element located downstream of the initiation codon (downstream box; DB) was investigated for its ability to enhance translation. Plasmids were constructed with a traditional Shine-Dalgarno sequence upstream of beta-galactosidase and one with the Shine-Dalgarno sequence and the DB within the coding sequence of the β-galactosidase. The proteins were translated in vitro with the E. coli S30 Extract System for Linear Templates. More β-galactosidase was expressed in the presence of the DB. (1180)

Notes: The gyrB gene of Bartonella bacilliformis isolated from a genomic library created in LambdaGEM®-11 (LambdaGEM®-11 BamH I Arms/Packagene® System). The authors used Promega's E.coli S30 Extract System to confirm that the E.coli transcription/translation machinery would recognize the B. bacilliformis gyrB sequence. (1469)

Notes: The ability of Shiga toxin to inhibit bacterial protein synthesis was assessed with the E. coli S30 Extract System for Linear Templates and a translatable poly U RNA. Translation reactions were incubated with increasing amounts of Shiga toxin, pokeweed antiviral protein or ricin A chain and then allowed to proceed for 5 minutes. Antibodies to the added proteins were used to remove them from the reaction and residual translation activity determined. Shiga toxin was a potent inhibitor of the ribosomes in the S30 extract. (0315)

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